Phosphate Biosensors and Methods of Using the Same

ABSTRACT

Phosphate biosensors are disclosed, which comprise a phosphate binding domain conjugated to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon phosphate binding. Such biosensors are useful for real time monitoring of phosphate metabolism in living cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to provisional application Ser. No.60/643,576, provisional application Ser. No. 60/658,141, provisionalapplication Ser. No. 60/658,142 and provisional application Ser. No.60/657,702, PCT application [Attorney Docket No. 056100-5055, “SucroseBiosensors and Methods of Using the Same”], and PCT application[Attorney Docket No. 056100-5054, “Methods of Reducing Repeat-InducedSilencing of Transgene Expression and Improved Fluorescent Biosensors”],which are herein incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This work was supported by Human Frontier Science Program grant No.RGP0041/2004C. The government may have certain rights to this invention.

FIELD OF INVENTION

The invention relates generally to the construction of phosphatebiosensors and methods for measuring and detecting changes in phosphatelevels using fluorescence resonance energy transfer (FRET).

BACKGROUND OF INVENTION

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

Phosphate (Pi) is an essential macronutrient for all living organisms.It is involved in most metabolic and signaling events in a cell, and ispresent in multiple cellular compartments. It serves various basicbiological functions as a structural element in nucleic acids,phospholipids and ATP, as a metabolite involved in energy transfer, as acomponent in signal transduction cascades, and in the regulation ofenzymes and metabolic processes.

Adenosine triphosphate (ATP) is the dominant ‘energy currency’ in thecell. The hydrolysis of ATP to adenosine diphosphate (ADP) plus Pireleases energy that fuels enumerable energy-requiring processes in thecell. Indeed, ATP is required for the phosphorylation of glucose (togenerate glucose 6-phosphate), which enables glucose to enter theglycolytic pathway. Complete aerobic oxidation of a single glucose6-phosphate molecule yields 30-36 molecules of ATP. Therefore, Pi is acritical metabolite and an essential nutrient, and the concentration ofthis molecule can profoundly alter cellular growth and metabolism. It issurprising, then, how little is known about the subcellular distributionphosphate and homeostasis under different phosphate concentrations.

To be able to measure phosphate levels directly in living cells, itwould be useful to have a nanosensor for phosphate. A phosphate sensorwould be an excellent tool for discovery and drug screening. Theresponse of phosphate levels could be measured in real time in responseto chemicals, metabolic events, transport steps, and signalingprocesses.

Recently a number of bacterial periplasmic binding proteins (PBP), whichundergo a venus flytrap-like closure of two lobes upon substratebinding, have been successfully used as the scaffold of metabolitenanosensors (Fehr, M., Frommer, W. B., and Lalonde, S. (2002)Visualization of maltose uptake in living yeast cells by fluorescentnanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851; Fehr, M.,Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivoimaging of the dynamics of glucose uptake in the cytosol of COS-7 cellsby fluorescent nanosensors. J. Biol. Chem. 278, 19127-19133; Lager, I.,Fehr, M., Frommer, W. B., and Lalonde, S. (2003) Development of afluorescent nanosensor for ribose. FEBS Lett 553, 85-89). The PBPnanosensors thus far developed have been constructed using type Iperiplasmic binding proteins, wherein the fluorophores attached to theN- and C-termini of the protein are located on two different lobes.

There is a PBP for phosphate (PiBP) that has been isolated from variousgram negative bacteria. For instance, the synthesis of the PiBP, theproduct of the pstS gene, is induced in E. coli when cell growth islimited by low Pi availability. However, in contrast to the type I PBPsused for nanosensors thus far, periplasmic phosphate binding protein hasbeen classified as a type II PBP, with N- and C-termini located on thesame protein lobe (Tam, R., and Saier, M. H. (1993) Microbiol Rev 57(2),320-346; Fukami-Kobayashi, K., Tateno, Y., and Nishikawa, K. (1999) JMol Biol 286(1), 279-290). The crystal structure of phosphate bindingprotein has been studied, and the modeled structures of PiBP alsosuggest a type II configuration although the assignment of both N- andC-terminal region is uncertain (Hirshberg, M., Henrick, K., Haire, L.L., Vasisht, N., Brune, M., Corrie, J. E. T., and Webb, M. R. (1998)Biochemistry-Us 37(29), 10381-10385; Ledvina, P. S., Tsai, A. L., Wang,Z. M., Koehl, E., and Quiocho, F. A. (1998) Protein Sci 7(12),2550-2559). In addition, phosphate quenches fluorescence, making theanalysis of phosphate sensors potentially problematic. Therefore, it wasnot clear whether a phosphate PBP sensor could be generated using thestrategies employed for type I PBP sensors.

SUMMARY OF INVENTION

The present inventors have surprisingly found that periplasmic phosphatebinding proteins may be used to construct biosensors for phosphate. Thepresent invention thus provides phosphate biosensors that may be usedfor detecting and measuring changes in phosphate concentrations inliving cells. In particular, the invention provides an isolated nucleicacid which encodes a phosphate fluorescent indicator, the indicatorcomprising a phosphate binding protein moiety, a donor fluorescentprotein moiety covalently coupled to the phosphate binding proteinmoiety, and an acceptor fluorescent protein moiety covalently coupled tothe phosphate binding protein moiety, wherein fluorescence resonanceenergy transfer (FRET) between the donor moiety and the acceptor moietyis altered when the donor moiety is excited and phosphate binds to thephosphate binding protein moiety. Vectors, including expression vectors,and host cells comprising the inventive nucleic acids are also provided,as well as biosensor proteins encoded by the nucleic acids. Such nucleicacids, vectors, host cells and proteins may be used in methods ofdetecting phosphate binding and changes in levels of phosphate, and inmethods of identifying compounds that modulate phosphate binding orphosphate-mediated activities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the predicted structure of Synechococcus PiBP and thealignment with E. coli PiBP (PDB 1a40) on both protein structure andbinding sites. (A) Phosphate-binding protein structure, predicted by3D-JIGSAW server and plotted by Deepview. The substrate phosphate islight and C- and N-terminus dark. (B) Alignment of the predicted proteinstructure with the crystal structure of E. coli. (C) Alignment ofbinding sites. The only difference is Glu70 on FLIPPi and ThriD on E.coli.

FIG. 2 shows graphs of in vitro substrate-induced FRET changes ofnanosensors purified from BL21(DE3) gold. (A) FLIPPi-WT titrated withphosphate solutions. (B) Phosphate titration curve for FLIPPi-260n. Thefitting curves are obtained by non-linear regression.

FIG. 3 is a graph showing in vitro substrate-induced FRET changes ofFLIPPi affinity mutants. The fitting curves were obtained by non-linearregression.

FIG. 4 contains graphs showing the results of binding specificity assaysfor the affinity mutants. All the sensors were titrated with solutionsof sulfate, nitrate, phosphate, pyrophosphate and ATP. (A) FLIPPi-30m.(B) FLIPPi-200-μ. (C) FLIPPi-5μ. (D) FLIPPi-4μ. (E) FLIPglu-6001μ ascontrol.

FIG. 5 contains graphs showing the results of further bindingspecificity assays for the affinity mutants. FLIPPi sensors weretitrated with glycerol-1-phosphate and glucose-6-phosphate solutions.(A) Titration with glycerol-1-phosphate. (B) Titration withglucose-6-phosphate.

FIG. 6 shows the FRET ratio change of FLIPPi-30m nanosensor in CHO cellsin response to 10 μM, 50 μM and 100 μM phosphate perfusions. Eachphosphate perfusion was 2 min, followed by 3 min wash with thephosphate-free modified Tyrode's saline solution. The numbers in the boxindicates the concentration of phosphate solution used.

FIG. 7 shows a phylogenic tree made by PAUP showing the relationshipbetween PiBP proteins of various species.

FIG. 8 is a diagram showing the deletions and the structure of theshortened FLIPPi-WT nanosensor containing deletions of material from theattached fluorophores.

DETAILED DESCRIPTION OF INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Other objects, advantages and features of the present invention becomeapparent to one skilled in the art upon reviewing the specification andthe drawings provided herein. Thus, further objects and advantages ofthe present invention will be clear from the description that follows.

Periplasmic Phosphate Binding Protein (PiBP)

The uptake of Pi into gram negative bacteria is initiated by the bindingof this anion to a periplasmically-localized, Pi-binding protein (PiBP).There are more than 20 known analogous binding proteins that function inthe uptake of sugars, oxyanions, amino acids, and oligopeptides. Thesynthesis of the PiBP, the product of the pstS gene, is induced in E.coli when cell growth is limited by low Pi availability. PiBP exhibitsspecific binding to both monobasic (H₂PO₄ ⁻) and dibasic (HPO₄ ²⁻)phosphate (Wang, Z. M., Choudhary, A., Ledvina, P. S., and Quiocho, F.A. (1994) J Biol Chem 269(40), 25091-25094), with a K_(d) of 0.8 nM(Medveczky, N., and Rosenberg, H. (1970) Biochim Biophys Acta 211(2),158). The dissociation rate constant is 21 s⁻¹ at pH 7.0 and low ionicstrength (Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R.(1994) Biochemistry-Us 33(27), 8262-8271).

The PiBP consists of two globular domains connected by peptide segmentsthat create a flexible hinge (Ledvina, P. S., Yao, N. H., Choudhary, A.,and Quiocho, F. A. (1996) P Natl Acad Sci USA 93(13), 6786-6791). In theabsence of Pi, the globular domains are separated, exposing a cleft thatis accessible to soluble metabolites. A conformational change in theprotein occurs upon binding of Pi, whereupon the globular domains comecloser together, the binding of Pi becomes tighter as a consequence ofhydrogen bonding of the Pi to amino acids of the cleft (Luecke, H., andQuiocho, F. A. (1990) Nature 347(6291), 402-406), and the binding pocketbecomes inaccessible to the solvent environment. As discussed above,PiBP has been classified as a type II PBP wherein the N- and C-terminiare located on the same lobe of the protein.

PiBP DNA sequences may be obtained from public databases, for instancethe NCBI website, or cloned from any Gram negative bacterium of interestusing techniques that are well known in the art. Cyanobacteria are agood source for PiBP sequences, as are thermophilic andhyperthermophilic bacteria, since the proteins isolated from thesebacteria display enhanced stability under conditions of extremetemperature, pH or chemical exposure. See Application Ser. No.60/658,142, which is herein incorporated by reference in its entirety. Aphylogenic tree made by PAUP, showing the relationship between PiBPsequences of various species, is shown in FIG. 7.

For instance, to exemplify the present invention, a truncated PiBP(protein accession NP_(—)415188), encoding the encoding the predictedmature protein without the signal sequence, was amplified by PCR from athermophilic strain of Synechococcus isolated from the hot springs ofYellowstone National Park. The sequence was amplified from genomic DNAusing the primers 5′-ATTGGTACCGTAGGATTTCTAACAGCG-3′ (SEQ ID NO: 1) and5′-ATAGGTACCGTTAACGGTGATGGAATC-3′ (SEQ ID NO: 2), and has the sequenceof SEQ ID NO: 3 including the signal sequence. The sequence of theSynechococcus sp. PiBP is 36.9% identical to PiBP of E. coli (encoded bypstS: PDB 1a40) (De Lorimier, R. M., Smith, J. J., Dwyer, M. A., Looger,L. L., Sali, K. M., Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D.W., Loew, L., and Hellinga, H. W. (2002) Protein Sci 11(11), 2655-2675),with the two proteins having a very similar predicted tertiary structure(see FIG. 1).

Biosensors

The present invention provides phosphate biosensors for detecting andmeasuring changes in phosphate concentrations using FluorescenceResonance Energy Transfer (FRET). The term “phosphate” includes bothmonobasic (H₂PO₄ ⁻) and dibasic (HPO₄ ²⁻) phosphate, and all other formsof phosphate for which a receptor exists.

In particular, the invention provides isolated nucleic acids encodingphosphate binding fluorescent indicators and the phosphate fluorescentindicators encoded thereby. One embodiment, among others, is an isolatednucleic acid which encodes a phosphate binding fluorescent indicator,the indicator comprising: a phosphate binding protein moiety, a donorfluorescent protein moiety covalently coupled to the phosphate bindingprotein moiety, and an acceptor fluorescent protein moiety covalentlycoupled to the phosphate binding protein moiety, wherein FRET betweenthe donor moiety and the acceptor moiety is altered when the donormoiety is excited and phosphate binds to the phosphate binding proteinmoiety.

As used herein, “covalently coupled” means that the donor and acceptorfluorescent moieties may be conjugated to the ligand binding proteinmoiety via a chemical linkage, for instance to a selected amino acid insaid ligand binding protein moiety. Covalently coupled also means thatthe donor and acceptor moieties may be genetically fused to the ligandbinding protein moiety such that the ligand binding protein moiety isexpressed as a fusion protein comprising the donor and acceptormoieties. As described herein, the donor and acceptor moieties may befused to the tennini of the phosphate binding moiety or to an internalposition within the phosphate binding moiety so long as FRET between thedonor moiety and the acceptor moiety is altered when the donor moiety isexcited and phosphate binds to the phosphate binding protein moiety.

A preferred phosphate binding protein moiety, among others, is aphosphate binding protein moiety from the Synechococcus PiBP proteinhaving the sequence of SEQ ID NO: 4. Any portion of the PiBP DNAsequence which encodes a phosphate binding region may be used in thenucleic acids of the present invention. Phosphate binding portions ofPiBP or any of its homologues from other organisms, for instance Gramnegative bacteria including thermophilic and hyperthermophilicorganisms, may be cloned into the vectors described herein and screenedfor activity according to the disclosed assays.

Naturally occurring species variants of PiBP may also be used, inaddition to artificially engineered variants comprising site-specificmutations, deletions or insertions that maintain measurable phosphatebinding function. Variant nucleic acid sequences suitable for use in thenucleic acid constructs of the present invention will preferably have atleast 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, or 99% similarity oridentity to the gene sequence for PiBP. Suitable variant nucleic acidsequences may also hybridize to the gene for PiBP under highly stringenthybridization conditions. High stringency conditions are known in theart; see for example Maniatis et al., Molecular Cloning: A LaboratoryManual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed.Ausubel, et al., both of which are hereby incorporated by reference.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993), whichis herein incorporated by reference. Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0M sodium ion, typically about 0.01to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

Preferred artificial variants of the present invention may be designedto exhibit decreased affinity for the ligand, in order to expand therange of ligand concentration that can be measured by the disclosednanosensors. Additional artificial variants showing decreased orincreased binding affinity for ligands may be constructed by random orsite-directed mutagenesis and other known mutagenesis techniques, andcloned into the vectors described herein and screened for activityaccording to the disclosed assays. The binding specificity of disclosedbiosensors may also be altered by mutagenesis so as to alter the ligandrecognized by the biosensor. See, for instance, Looger et al., Nature,423 (6936): 185-190. Due to the similarity of sulfate and phosphatePBPs, it may also be possible to create a phosphate binding protein froma sulfate binding protein using site-directed mutagenesis.

The sensors of the invention may also be designed with a phosphatebinding moiety and one or more additional protein binding moieties thatare covalently coupled or fused together and to the donor and acceptorfluorescent moieties in order to generate an allosteric enzyme whoseactivity is controlled by more than one ligand. Allosteric enzymescontaining dual specificity for more than one ligand have been describedin the art, and may be used to construct the FRET biosensors describedherein (Guntas and Ostermeier, 2004, J. Mol. Biol. 336(1): 263-73).

The isolated nucleic acids of the invention may incorporate any suitabledonor and acceptor fluorescent protein moieties that are capable incombination of serving as donor and acceptor moieties in FRET. Preferreddonor and acceptor moieties are selected from the group consisting ofGFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP(blue fluorescent protein), YFP (yellow fluorescent protein), andenhanced variants thereof, with a particularly preferred embodimentprovided by the donor/acceptor pair CFP/YFP Venus, a variant of YFP withimproved pH tolerance and maturation time (Nagai, T., Ibata, K., Park,E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant ofyellow fluorescent protein with fast and efficient maturation forcell-biological applications. Nat. Biotechnol. 20, 87-90). Analternative is the MiCy/mKO pair with higher pH stability and a largerspectral separation (Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A.Cyan-emitting and orange-emitting fluorescent proteins as adonor/acceptor pair for fluorescence resonance energy transfer. BiochemJ. 2004 381:307-12). Also suitable as either a donor or acceptor isnative DsRed from a Discosoma species, an ortholog of DsRed from anothergenus, or a variant of a native DsRed with optimized properties (e.g. aK83M variant or DsRed2 (available from Clontech)). Criteria to considerwhen selecting donor and acceptor fluorescent moieties is known in theart, for instance as disclosed in U.S. Pat. No. 6,197,928, which isherein incorporated by reference in its entirety.

As used herein, the term “variant” is intended to refer to polypeptideswith at least about 30%, 40%, 50%, 60%, 70%, more preferably at least75% identity, including at least 80%, 90%, 95% or greater identity tonative fluorescent molecules. Many such variants are known in the art,or can be readily prepared by random or directed mutagenesis of a nativefluorescent molecules (see, for example, Fradkov et al., FEBS Lett.479:127-130 (2000)).

When the fluorophores of the biosensor contain stretches of similar orrelated sequence(s), the present inventors have recently discovered thatgene silencing may adversely affect expression of the biosensor incertain cells and particularly whole organisms. In such instances, it ispossible to modify the fluorophore coding sequences at one or moredegenerate or wobble positions of the codons of each fluorophore, suchthat the nucleic acid sequences of the fluorophores are modified but notthe encoded amino acid sequences. Alternative, one or more conservativesubstitutions that do not adversely affect the function of thefluorophores may also be incorporated. See PCT application [AttorneyDocket No. 056100-5054, “Methods of Reducing Repeat-Induced Silencing ofTransgene Expression and Improved Fluorescent Biosensors], which isherein incorporated by reference in its entirety.

It is also possible to use or luminescent quantum dots (QD) for FRET(Clapp et al., 2005, J. Am. Chem. Soc. 127(4): 1242-50), dyes, includingbut not limited to TOTO dyes (Laib and Seeger, 2004, J. Fluoresc.14(2):187-91), Cy3 and Cy5 (Churchman et al., 2005, Proc Natl Acad SciUSA. 102(5): 1419-23), Texas Red, fluorescein, and tetramethylrhodamine(TAMRA) (Unruh et al., Photochem Photobiol. 2004 Oct. 1), AlexaFluor488, to name a few, as well as fluorescent tags (see, for example,Hoffman et al., 2005, Nat. Methods 2(3): 171-76).

The invention further provides vectors containing isolated nucleic acidmolecules encoding the biosensor polypeptides described herein.Exemplary vectors include vectors derived from a virus, such as abacteriophage, a baculovirus or a retrovirus, and vectors derived frombacteria or a combination of bacterial sequences and sequences fromother organisms, such as a cosmid or a plasmid. Such vectors includeexpression vectors containing expression control sequences operativelylinked to the nucleic acid sequence coding for the biosensor. Vectorsmay be adapted for function in a prokaryotic cell, such as E. coli orother bacteria, or a eukaryotic cell, including animal cells or plantcells. For instance, the vectors of the invention will generally containelements such as an origin of replication compatible with the intendedhost cells, one or more selectable markers compatible with the intendedhost cells and one or more multiple cloning sites. The choice ofparticular elements to include in a vector will depend on factors suchas the intended host cells, the insert size, whether regulatedexpression of the inserted sequence is desired, i.e., for instancethrough the use of an inducible or regulatable promoter, the desiredcopy number of the vector, the desired selection system, and the like.The factors involved in ensuring compatibility between a host cell and avector for different applications are well known in the art.

Preferred vectors for use in the present invention will permit cloningof the phosphate binding domain or receptor between nucleic acidsencoding donor and acceptor fluorescent molecules, resulting inexpression of a chimeric or fusion protein comprising the phosphatebinding domain covalently coupled to donor and acceptor fluorescentmolecules. Exemplary vectors include the bacterial pRSET-FLIPderivatives disclosed in Fehr et al. (2002) (Visualization of maltoseuptake in living yeast cells by fluorescent nanosensors, Proc. Natl.Acad. Sci. USA 99, 9846-9851), which is herein incorporated by referencein its entirety. Methods of cloning nucleic acids into vectors in thecorrect frame so as to express a fusion protein are well known in theart.

The phosphate biosensors of the present invention may be expressed inany location in the cell, including the cytoplasm, cell surface orsubcellular organelles such as the nucleus, vesicles, ER, vacuole, etc.Methods and vector components for targeting the expression of proteinsto different cellular compartments are well known in the art, with thechoice dependent on the particular cell or organism in which thebiosensor is expressed. See, for instance, Okumoto, S., Looger, L. L.,Micheva, K. D., Reimer, R. J., Smith, S. J., and Frommer, W. B. (2005) PNatl Acad Sci USA 102(24), 8740-8745; Fehr, M., Lalonde, S., Ehrhardt,D. W., and Frommer, W. B. (2004) J Fluoresc 14(5), 603-609, which areherein incorporated by reference in their entireties.

The chimeric nucleic acids of the present invention may be constructedsuch that the donor and acceptor fluorescent moiety coding sequences arefused to separate termini of the ligand binding domain in a manner suchthat changes in FRET between donor and acceptor may be detected uponligand binding. Fluorescent domains can optionally be separated from theligand binding domain by one or more flexible linker sequences. Suchlinker moieties are preferably between about 1 and 50 amino acidresidues in length, and more preferably between about 1 and 30 aminoacid residues. Linker moieties and their applications are well known inthe art and described, for example, in U.S. Pat. Nos. 5,998,204 and5,981,200, and Newton et al., Biochemistry 35:545-553 (1996).Alternatively, shortened versions of linkers or any of the fluorophoresdescribed herein may be used. For example, the inventors have shown thatdeleting N- or C-terminal portions of any of the three modules can leadto increased FRET ratio changes, as described in Application Ser. No.60/658,141, which is herein incorporated by reference in its entirety.

It will also be possible depending on the nature and size of thephosphate binding domain to insert one or both of the fluorescentmolecule coding sequences within the open reading frame of the phosphatebinding protein such that the fluorescent moieties are expressed anddisplayed from a location within the biosensor rather than at thetermini. Such sensors are generally described in U.S. Application Ser.No. 60/658,141, which is herein incorporated by reference in itsentirety. It will also be possible to insert a phosphate bindingsequence into a single fluorophore coding sequence, i.e. a sequenceencoding a GFP, YFP, CFP, BFP, etc., rather than between tandemmolecules. According to the disclosures of U.S. Pat. No. 6,469,154 andU.S. Pat. No. 6,783,958, each of which is incorporated herein byreference in their entirety, such sensors respond by producingdetectable changes within the protein that influence the activity of thefluorophore.

The invention also includes host cells transfected with a vector or anexpression vector of the invention, including prokaryotic cells, such asE. coli or other bacteria, or eukaryotic cells, such as yeast cells,animal cells or plant cells. In another aspect, the invention features atransgenic non-human animal having a phenotype characterized byexpression of the nucleic acid sequence coding for the expression of theenvironmentally stable biosensor. The phenotype is conferred by atransgene contained in the somatic and germ cells of the animal, whichmay be produced by (a) introducing a transgene into a zygote of ananimal, the transgene comprising a DNA construct encoding the phosphatebiosensor; (b) transplanting the zygote into a pseudopregnant animal;(c) allowing the zygote to develop to term; and (d) identifying at leastone transgenic offspring containing the transgene. The step ofintroducing of the transgene into the embryo can be achieved byintroducing an embryonic stem cell containing the transgene into theembryo, or infecting the embryo with a retrovirus containing thetransgene. Transgenic animals of the invention include transgenic C.elegans and transgenic mice and other animals. Transgenic plants arealso included.

The present invention also encompasses isolated phosphate biosensormolecules having the properties described herein, particularly phosphatebinding fluorescent indicators constructed using hyperthermophilic andmoderately thermophilic proteins. Such polypeptides may be recombinantlyexpressed using the nucleic acid constructs described herein, orproduced by chemically coupling some or all of the component domains.The expressed polypeptides can optionally be produced in and/or isolatedfrom a transcription-translation system or from a recombinant cell, bybiochemical and/or immunological purification methods known in the art.The polypeptides of the invention can be introduced into a lipidbilayer, such as a cellular membrane extract, or an artificial lipidbilayer (e.g. a liposome vesicle) or nanoparticle.

Methods of Detecting Phosphate

The nucleic acids and proteins of the present invention are useful fordetecting phosphate binding and measuring changes in the levels ofphosphate both in vitro and in a plant or an animal. In one embodiment,the invention comprises a method of detecting changes in the level ofphosphate in a sample of cells, comprising (a) providing a cellexpressing a nucleic acid encoding a phosphate biosensor as describedherein and a sample of cells; and (b) detecting a change in FRET betweena donor fluorescent protein moiety and an acceptor fluorescent proteinmoiety, each covalently attached to the phosphate binding domain,wherein a change in FRET between said donor moiety and said acceptormoiety indicates a change in the level of phosphate in the sample ofcells.

FRET may be measured using a variety of techniques known in the art. Forinstance, the step of determining FRET may comprise measuring lightemitted from the acceptor fluorescent protein moiety. Alternatively, thestep of determining FRET may comprise measuring light emitted from thedonor fluorescent protein moiety, measuring light emitted from theacceptor fluorescent protein moiety, and calculating a ratio of thelight emitted from the donor fluorescent protein moiety and the lightemitted from the acceptor fluorescent protein moiety. The step ofdetermining FRET may also comprise measuring the excited state lifetimeof the donor moiety or anisotropy changes (Squire A, Verveer P J, RocksO, Bastiaens P I. J Struct Biol. 2004 July; 147(1):62-9. Red-edgeanisotropy microscopy enables dynamic imaging of homo-FRET between greenfluorescent proteins in cells.). Such methods are known in the art anddescribed generally in U.S. Pat. No. 6,197,928, which is hereinincorporated by reference in its entirety.

The amount of phosphate in a sample of cells can be determined bydetermining the degree of FRET. First the sensor must be introduced intothe sample. Changes in phosphate concentration can be determined bymonitoring FRET at a first and second time after contact between thesample and the fluorescent indicator and determining the difference inthe degree of FRET. The amount of phosphate in the sample can bequantified for example by using a calibration curve established bytitration.

The cell sample to be analyzed by the methods of the invention may becontained in vivo, for instance in the measurement of phosphatetransport or signaling on the surface of cells, or in vitro, whereinphosphate efflux may be measured in cell culture. Alternatively, a fluidextract from cells or tissues may be used as a sample from whichphosphate is detected or measured.

Methods for detecting phosphate levels as disclosed herein may be usedto screen and identify compounds that may be used to modulate phosphateconcentrations and activities relating to phosphate changes. In oneembodiment, among others, the invention comprises a method ofidentifying a compound that modulates phosphate binding or levelscomprising (a) contacting a mixture comprising a cell expressing anphosphate biosensor as disclosed herein and a sample of cells with oneor more test compounds, and (b) determining FRET between said donorfluorescent domain and said acceptor fluorescent domain following saidcontacting, wherein increased or decreased FRET following saidcontacting indicates that said test compound is a compound thatmodulates phosphate binding activity or phosphate levels.

The term “modulate” in this embodiment means that such compounds mayincrease or decrease phosphate binding activity, or may affectactivities, i.e., cell functions or signaling cascades, that affectphosphate levels. Compounds that increase or decrease phosphate bindingactivity may be targets for therapeutic intervention and treatment ofdisorders associated with aberrant phosphate activity, or with aberrantcell metabolism or signal transduction, as described above. Othercompounds that increase or decrease phosphate binding activity orphosphate levels associated with cellular functions may be developedinto therapeutic products for the treatment of disorders associated withligand binding activity.

Utilities

The phosphate sensors of the present invention will be useful for a widerange of applications, e.g. to study phosphate levels in marine systemswith better precision. New tools for such measurements are requiredsince the marine biogeochemical phosphorus cycle is linked to carbonfluxes and the partitioning of both major and minor elements in theocean. The sensors will be useful to study the biochemical pathways invivo, i.e., to determine phosphate flux in microorganisms, in soil andalso in eukaryotes. Metabolism is regulated in response to variations inphosphate supply since many reactions are coupled to phosphorylation.Thus this sensor provides a new tool to characterize phosphatehomeostasis in healthy and diseased conditions. It can be used as a toolto develop new chemicals that positively or negatively affect phosphatehomeostasis in high throughput screens. It can be used to characterizecellular uptake and release, and more importantly intracellularcompartmentation. At present, e.g. phosphate exchange between ER andcytosol of liver cells is not understood. The sensors can be used, tostudy phosphate homeostasis in the ER during hepatocytes glucosemetabolism, storage and mobilization phases. The sensors can be used tocharacterize the unusual link between phosphate and glutamate transportin VGLUT transporters in neurons and may help elucidating importantaspects of neurotransmission in healthy and diseased conditions, i.e.the link between glutamate secretion and phosphate uptake and thus maybe of relevance for all diseases linked to glutamergic transmission.

It is important for an organism to maintain inorganic phosphatehomeostasis. An excess or deficiency of phosphate may each cause variousdisorders. Renal disease can lead to loss of phosphate, resulting indiseases such as X-linked hypophosphatemia (XLH), autosomal dominanthypophosphatemic rickets (ADHR), hereditary hypophosphatemic ricketswith hypercalciuria (HHRH) and oncogenic hypophosphatemic osteomalacia(OHO) (Tenenhouse and Murer, 2003, J. Am. Soc. Nephrol. 14: 240-247).This is because the kidney plays a major role in clearing and retaininginorganic phosphate in the body. Physiological studies have shown thatthe proximal tubule reabsorbs a bulk of the phosphate filtered throughthe kidney. Phosphate reabsorption in the proximal tubule is mediated byphosphate transporters. Malfunction of these phosphate transportersleads to the inhibition of phosphate reabsorption, which in turn leansto phosphate wasting.

On the other hand, elevated levels of the mineral phosphate may signalan increased risk of death for patients with chronic kidney disease. Arecent study has shown that elevated phosphate levels are associatedwith increased mortality risk in chronic kidney disease (Kestenbaum etal. 2005, J. Am. Soc. Nephrol., 16: 520-528). Therefore, elevated levelsof phosphate can be considered as an important danger sign in chronickidney disease. Phosphate toxicity can occur when laxatives or enemasthat contain phosphate are used in high doses. For all these and othercases, the sensors may provide tools to investigate the underlyingdefects and to develop cures.

Phosphate, as for many microorganisms, is an essential macronutrient forplant cells. The sensors may provide a means to determine the soilphosphate levels in the filed as well as the fertilization status of theplant. The sensor will help in the elucidation of mechanisms forphosphate homeostasis in plants and help in the design of improved cropswith better phosphate efficiency.

Transgenic organisms expressing FLIPPi can be used to detect phosphateactivity directly in organ slices or whole organisms as demonstrated forthe calcium FRET indicator in Caenorhabditis elegans neurons (Kerr, R.,Lev-Ram, V., Baird, G., Vincent, P., Tsien, R. Y., and Schafer, W. R.(2000) Neuron 26(3), 583-594). The phosphate sensors of the presentinvention are excellent tools for drug discovery and screening.Phosphate levels may be measured in real time in response to chemicals,metabolic events, transport steps and signaling processes.

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

EXAMPLES Example 1 Cloning and Structural Modeling of PiBP

The bacterial PiBP functions as a high-affinity in vitro Pi sensor whencoupled to environmentally-sensitive small fluorophore dyes (DeLorimier, R. M., Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M.,Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., andHelling a, H. W. (2002) Protein Sci 11(11), 2655-2675). Therefore, wechose to use this polypeptide to construct a genetically-encodednanosensor capable of reporting changes in the intracellularconcentrations of Pi. Given that proteins from thermophilic organismsshow enhanced stability under harsh environmental conditions andconceivably provide a more robust framework for nanosensor construction(see Application Ser. No. 60/658,142, which is herein incorporated byreference), we chose a thermophilic strain of Synechococcus isolatedfrom the hot springs of Yellowstone National Park as the source forPiBP.

To clone the PiBP gene from Synechococcus, a truncated PiBP (proteinaccession NP_(—)415188), encoding the predicted mature protein withoutthe signal sequence, was amplified by PCR from Synechococcus genomic DNAusing the primers 5′-ATTGGTACCGTAGGATTTCTAACAGCG-3′ (SEQ ID NO: 1) and5′-ATAGGTACCGTTAACGG TGATGGAATC-3′ (SEQ ID NO: 2). The PCR fragment wascloned into the KpnI site of FLIPmal-25μ (Fehr, M., Frommer, W. B., andLalonde, S. (2002) P Natl Acad Sci USA 99(15), 9846-9851) in pRSET-B(Invitrogen, USA), exchanging the sequence encoding the maltose-bindingprotein with that of PiBP. The resulting plasmid was namedpRSET-FLIPPi-250n.

To improve maturation proficiency and the performance of the sensor(with respect to pH and chloride tolerance), enhanced YFP inpRSET-FLIPPi-250n was replaced with the coding sequence of the YFPvariant designated Venus (forming FLIPPi-WT) (SEQ ID NO: 5 (gene) andSEQ ID NO: 6 (protein)) (Nagai, T., Ibata, K., Park, E. S., Kubota, M.,Mikoshiba, K., and Miyawaki, A. (2002) Nat Biotechnol 20(1), 87-90).Affinity mutants (verified by sequencing the mutant DNA fragments) forFLIPPi were created by site-directed mutagenesis (Kunkel, T. A.,Roberts, J. D., and Zakour, R. A. (1987) Methods in Enzymology 154,367-382), generating the T22A, S52A, G162A and T163A variant sensorproteins. These pRSET-FLIPPi constructs were introduced into E. coliBL21(DE3) Gold (Stratagene, USA) by electroporation and the expressedproteins were extracted and purified as described (Fehr, M., Frommer, W.B., and Lalonde, S. (2002) P Natl Acad Sci USA 99(15), 9846-9851). Forexpression in the cytosol of CHO cells, DNA fragments containingFLIPPi-5μ and FLIPPi-30m sequences were excised from pRSET-FLIPPi-5μ andpRSET-FLIPPi-30m with BamHI/HindIII and cloned into pcDNA3.1(Invitrogen, USA).

The sequence of the Synechococcus sp. PiBP was aligned with PiBP of E.coli (encoded by pstS: PDB 1a40, identity 36.9%) (De Lorimier, R. M.,Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M., Paavola, C. D.,Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., and Helling a, H. W.(2002) Protein Sci 11(11), 2655-2675), and the putative periplasmicleader sequence was removed from the sequence prior to structuralprediction analyses. The Synechococcus sp. PiBP structure was modeledusing the automated prediction algorithm resident on the 3D-JIGSAWserver. FIG. 1 shows the predicted polypeptide structure (A), along withthe Deepview v3.7SP5 alignment of this structure with that of E. coliPiBP (B). The tertiary structure shown is similar to that of the E. coliPiBP, which provided a sound foundation for sensor construct.

Example 2 In Vitro Characterization of Nanosensors

A DNA fragment encoding the mature Synechococcus sp. PiBP protein wasfused between the ECFP and Venus sequences as described above. Thechimeric gene was expressed in E. coli and the protein product purifiedvia the N-terminal His₆ tag using Ni²⁺ affinity chromatography.

Addition of Pi to the purified protein resulted in a decrease in CFP andan increase in Venus emission, suggesting that binding of Pi to thechimeric sensor, designated FLIPPi-WT, resulted in a conformationalchange that changed the distance/orientation of the chromophores on theengineered protein. The binding constant (K_(d)) of this sensor for Piwas determined to be 840 nM and the Hill coefficient was 1.03, which isconsistent with 800 nM K_(d) as reported for the E. coli PiBP byMedveczky et al. (Medveczky, N., and Rosenberg, H. (1970) BiochimBiophys Acta 211(2), 158). As shown in FIG. 2A, the maximum ratio changeobserved for FLIPPi-WT is 0.14.

In attempts to modify the properties of the FLIPPi-WT nanosensor, 9amino acids were deleted from the C-terminus of the CFP and 1 amino acidwas deleted from the N-terminus of VENUS. Furthermore, four amino acidsin the linker domain attaching the fluorophores to the Pi-binding domainwere also removed. In total, the protein was shorted by 18 amino acids.The deletions and the structure of the shortened polypeptide are shownin FIG. 8. As shown in FIG. 3, titration of the modified protein,designated FLIPPi-260 nm, with Pi caused a CFP emission increase andVenus emission decrease, resulting in a Pi-dependant decrease in FRET.The binding constant for Pi was determined as 260 nM, with a maximumratio change of 0.13. This change was slightly reduced relative to thatof the original FLIPPi-WT fusion protein.

To expand the dynamic range of the Pi sensor, site directed mutagenesiswas used as described above to lower the binding affinity of the Pibinding domain. The Pi present in the binding pocket of PiBP is held inplace by strong hydrogen bonds (Luecke, H., and Quiocho, F. A. (1990)Nature 347(6291), 402-406). The three dimensional spatial structure ofthe ligand-binding pocket of FLIPPi-260n was aligned to the E. coli PiBPcrystal structure, PDB 1a40 (Wang, Z. M., Choudhary, A., Ledvina, P. S.,and Quiocho, F. A. (1994) J Biol Chem 269(40), 25091-25094; Ledvina, P.S., Tsai, A. L., Wang, Z. M., Koehl, E., and Quiocho, F. A. (1998)Protein Sci 7(12), 2550-2559). The analysis showed that the Pi bindingsite of FLIPPi-260n is very similar to that of E. coli except that theconserved Asp56 of the E. coli protein corresponds to Glu70 ofFLIPPi-260n (FIG. 1C). Asp56 of the E. coli protein is considered toplay a key role in recognizing both monobasic and dibasic Pi (Wang, Z.M., Choudhary, A., Ledvina, P. S., and Quiocho, F. A. (1994) J Biol Chem269(40), 25091-25094).

Five residues of FLIPPi-260n were selected for site-directedmutagenesis. Mutation of amino acids predicted to reside in thePi-binding site of FLIPPi-260n generated a set of sensors that weresensitive over a broad range of Pi concentrations, with significantlygreater maximum ratio changes upon Pi binding (see Table 1). Theaffinity mutants were designated FLIPPi-770n, FLIPPi-4μ, FLIPPi-5μ,FLIPPi-200μ, and FLIPPi-30m, according to their K_(d) for Pi. The Hillcoefficient for all of these PiBPs was determined to be close to 1,which agrees with that reported by Medveczky et al. and indicates thatthere is no cooperativity in substrate binding (Medveczky, N., andRosenberg, H. (1970) Biochim Biophys Acta 211(2), 158).

TABLE 1 FLIPPi affinity mutants. Binding constants determined in vitro.Name of Mutation Hill Range for R² sensor form K_(d) (M) ΔR_(max) ^(a)coefficient quantification^(b) (n ≧ 3) FLIPPi-260n Wild 2.6 × 10⁻⁷ −0.131.03 25.9-1260 nM 0.9953 type FLIPPi-770n S161A 7.7 × 10⁻⁷ −1.07 1.0385.7-5650 nM 0.9989 FLIPPi-4μ T163A 3.9 × 10⁻⁶ −1.34 1.02 0.382-25.2 μM0.9989 FLIPPi-5μ S52A 5.1 × 10⁻⁶ −1.33 1.03 0.516-34.0 μM 0.9994FLIPPi-200μ G162A 2.1 × 10⁻⁴ −1.13 1.00 252-1660 μM 0.9991 FLIPPi-30mT22A 0.033 −1.03 1.03 3.08-169 mM 0.9882 ^(a)ΔR_(max,) in vitro maximumchange in ratio between absence and saturation of the binding protein^(b)Range of concentration for which a FLIPPi sensor can be used. Therange for quantification was defined as the range between 10% and 90%saturation.

Each PiBP binds one molecule of Pi. Introducing substitution S161A intothe PiBP moiety of FLIPPi-260n yielded FLIPPi-770n, which has a bindingconstant for Pi of 770 nM and can be used for quantifying Pi levels thatrange 0.0847 and 5.65 μM. FLIPPi-770n had a 4-fold wider measuring rangeand an 8-fold increase in the maximum ratio change (ΔR_(max)=−1.07)relative to those of the FLIPPi-260n polypeptide (range of 25.9-1260 nMand ΔR_(max)=−0.13). All the affinity mutants that were examined(Table 1) had maximum ratio change improvement of 8-10 fold. FLIPPi-4μand FLIPPi-5μ, carrying the substitutions of T163A and S52A,respectively, had similar K_(d) (3.9 μM for FLIPPi-4μ and 5.1 μM forFLIPPi-5μ), ΔR_(max) (−1.34 and 1.33), and quantification range (0.382to 25.2 μM and 0.516 to 34.0 μM). FLIPPi-200μ (G162A substitution) issuitable for Pi quantification in the micromolar range (252 to 1660 μM,K_(d)=210 μM). The sensor with the best range for monitoring in vivo Piconcentrations is FLIPPi-30m. This sensor can be used over the range of3.08 to 169 mM, Pi, which generally spans the intracellularconcentration.

Example 3 Substrate Binding Specificity

Measuring substrate concentration in complex mixtures (e.g. cytoplasm ofa living cell) requires sensors with high specificity towards theirsubstrate. Therefore, for in vivo applications, it is necessary to testthe binding specificity of each of the sensors.

FRET characteristics of FLIPPi-4μ, FLIPPi-5μ, FLIPPi-200μ, andFLIPPi-30m were monitored at different concentrations of Pi and withpotential nonspecific substrates, including sodium sulfate, sodiumnitrate, ATP, sodium pyrophosphate. The FRET assays were performed inmicrotitre plates (FIG. 4A-D), with the glucose sensor FLIPglu-600μ(Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B.(2003) J Biol Chem 278(21), 19127-19133) used as a control (FIG. 4E).

FLIPPi-30m, FLIPPi-200μ and the control FLIPglu-600μ did not bind thefour nonspecific substrates. FLIPPi-4μ and FLIPPi-5μ responded topyrophosphate, with lower binding constants than those for Pi, whilethese sensors did not respond to the other three substrates. ATP seemedto be a quencher to all FLIPPi sensors tested, and pyrophosphate wasanother quencher to FLIPPi-30m and FLIPPi-200μ. Glycerol-1-phosphate andglucose-6-phosphate were also used for specificity tests with FLIPPi-4μ,FLIPPi-5μ, FLIPPi-200μ, and FLIPPi-30m together with the wild typeFLIPPi-260n (FIG. 5). The results demonstrate that while FLIPPi-200-μ,FLIPPi-30m with FLIPPi-260n did not respond to either substrate, thesesubstrates did affect the fluorescence emission ratios for FLIPPi-4μ andFLIPPi-5μ.

Example 4 In Vivo Characterization of Nanosensors

To demonstrate in vivo application of FLIPPi sensors, pRSET-FLIPPi-30mand pRSET-FLIPPi-5μ sequences were cloned in pcDNA3.1 and transfectedinto CHO cells. The CHO cells were grown overnight after transfectionand then starved for Pi for 16 h by replacing the Pi-replete growthmedium with modified Tyrode's saline solution containing no Pi. Cellsexpressing FLIPPi-30m were perfused with increasing concentrations ofexternal Pi along a specific step schedule (FIG. 6) and changes in thefluorescence ratio were monitored. Furthermore, after a constantfluorescence ratio was attained at each Pi concentration, the Pi wasremoved by perfusion with Pi-free solution. Cells transfected withFLIPPi-5μ was used as control because of its high affinity for Pi.

Cells grown under normal growth conditions, no matter which FLIPPisensor was expressed, did not show a signal change upon perfusion withup to 10 mM Pi (data not shown). Starved cells expressing FLIPPi-30mresponded to solutions containing 10 μM, 50 μM and 100 μM Pi; thehighest ratio change was for 100 μM Pi ratio changes (FIG. 6). Starvedcontrol cells did not show any VENUS/CFP ratio change when perfused withsame Pi solutions.

All publications, patents and patent applications discussed herein areincorporated herein by reference. While the invention has been describedin connection with specific embodiments thereof, it will be understoodthat it is capable of further modifications and this application isintended to cover any variations, uses, or adaptations of the inventionfollowing, in general, the principles of the invention and includingsuch departures from the present disclosure as come within known orcustomary practice within the art to which the invention pertains and asmay be applied to the essential features hereinbefore set forth and asfollows in the scope of the appended claims.

1. An isolated nucleic acid which encodes a phosphate fluorescentindicator, the indicator comprising: a phosphate binding protein moiety;a donor fluorescent protein moiety covalently coupled to the phosphatebinding protein moiety; and an acceptor fluorescent protein moietycovalently coupled to the phosphate binding protein moiety; whereinfluorescence resonance energy transfer (FRET) between the donor moietyand the acceptor moiety is altered when the donor moiety is excited andphosphate binds to the phosphate binding protein moiety.
 2. The isolatednucleic acid of claim 1, wherein the donor and acceptor moieties aregenetically fused to said phosphate binding protein moiety.
 3. Theisolated nucleic acid of claim 2, wherein the donor and acceptormoieties are genetically fused to the termini of said phosphate bindingprotein moiety.
 4. The isolated nucleic acid of claim 2, wherein one orboth the donor and acceptor moieties are genetically fused to aninternal position of said phosphate binding protein moiety.
 5. Theisolated nucleic acid of claim 1, wherein said phosphate binding proteinmoiety is a bacterial periplasmic binding protein (PBP) moiety.
 6. Theisolated nucleic acid of claim 1, wherein said PBP is the product of abacterial phoS gene.
 7. The isolated nucleic acid of claim 1, whereinsaid phosphate binding protein moiety is from a thermophilicmicroorganism.
 8. The isolated nucleic acid of claim 7, wherein saidthermophilic microorganism is an extreme thermophile or a moderatethermophile.
 9. The isolated nucleic acid of claim 7, wherein saidmicroorganism is a thermophilic species of Synechococcus.
 10. Theisolated nucleic acid of claim 9, wherein said phosphate binding proteinis PiBP from Synechococcus.
 11. The isolated nucleic acid of claim 10,wherein said phosphate binding protein moiety comprises the sequence ofSEQ ID No.
 1. 12. The isolated nucleic acid of claim 1, wherein saiddonor fluorescent protein moiety is selected from the group consistingof a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy)and monomeric CoralHue Kusabira-Orange (mKO).
 13. The isolated nucleicacid of claim 1, wherein said acceptor fluorescent protein moiety isselected from the group consisting of a GFP, a CFP, a BFP, a YFP, adsRED, CoralHue Midoriishi-Cyan (MiCy) and monomeric CoralHueKusabira-Orange (mKO).
 14. The isolated nucleic acid of claim 1, whereinsaid donor fluorescent protein moiety is a CFP and said acceptorfluorescent protein moiety is YFP Venus.
 15. The isolated nucleic acidof claim 1, further comprising at least one linker moiety.
 16. Theisolated nucleic acid of claim 15, further comprising a deletion,insertion or mutation of one or more amino acids in said phosphatebinding protein moiety, said donor fluorescent moiety, said acceptorfluorescent moiety and/or said at least one linker.
 17. The isolatednucleic acid of claim 16, wherein said phosphate fluorescent indicatorshows increased or decreased affinity for phosphate.
 18. The isolatednucleic acid of claim 16, wherein said phosphate fluorescent indicatorshows an increase in maximum FRET ratio change.
 19. A cell expressingthe nucleic acid of claim
 1. 20. The cell of claim 19, wherein thephosphate fluorescent sensor is expressed in the cytosol of said cell.21. The cell of claim 19, wherein the phosphate fluorescent sensor isexpressed on the surface of said cell.
 22. The cell of claim 19, whereinthe phosphate fluorescent sensor is expressed in the nucleus of saidcell.
 23. The cell of claim 19, wherein the cell is a prokaryote. 24.The cell of claim 19, wherein the cell is a eukaryotic cell.
 25. Thecell of claim 24, wherein the cell is a yeast cell.
 26. The cell ofclaim 24, wherein the cell is an animal cell.
 27. An expression vectorcomprising the nucleic acid of claim
 1. 28. A cell comprising the vectorof claim
 27. 29. The expression vector of claim 27 adapted for functionin a prokaryotic cell.
 30. The expression vector of claim 27 adapted forfunction in a eukaryotic cell.
 31. A transgenic animal expressing thenucleic acid of claim
 1. 32. The transgenic animal of claim 31, whereinsaid transgenic animal is C. elegans.
 33. A phosphate bindingfluorescent indicator encoded by the nucleic acid of claim
 1. 34. Amethod of detecting changes in the level of phosphate in a sample ofcells, comprising: (a) providing a cell expressing the nucleic acid ofclaim 1; and (b) detecting a change in FRET between said donorfluorescent protein moiety and said acceptor fluorescent protein moiety,wherein a change in FRET between said donor moiety and said acceptormoiety indicates a change in the level of phosphate in a sample ofcells.
 35. The method of claim 34, wherein the step of determining FRETcomprises measuring light emitted from the acceptor fluorescent proteinmoiety.
 36. The method of claim 34, wherein determining FRET comprisesmeasuring light emitted from the donor fluorescent protein moiety,measuring light emitted from the acceptor fluorescent protein moiety,and calculating a ratio of the light emitted from the donor fluorescentprotein moiety and the light emitted from the acceptor fluorescentprotein moiety.
 37. The method of claim 34, wherein the step ofdetermining FRET comprises measuring the excited state lifetime of thedonor moiety.
 38. The method of claim 34, wherein said sample of cellsis contained in vivo.
 39. The method of claim 34, wherein said sample ofcells is contained in vitro.
 40. A method of identifying a compound thatmodulates the binding of a phosphate to its receptor, comprising: (a)contacting a cell expressing the nucleic acid of claim 1 with one ormore test compounds in the presence of phosphate; and (b) determiningFRET between said donor fluorescent domain and said acceptor fluorescentdomain following said contacting, wherein increased or decreased FRETfollowing said contacting indicates that said test compound is acompound that modulates phosphate binding.